In Vitro Screening for Drought Tolerance in Different ... · In Vitro Screening for Drought Tolerance... ORIGINAL ARTICLE In Vitro Screening for Drought Tolerance in Different Sorghum

Journal of Stress Physiology & Biochemistry, Vol. 9 No. 3 2013, pp. 72-83 ISSN 1997-0838Original Text Copyright © 2013 by Tsago, Andargie, Takele

ORIGINAL ARTICLE

In Vitro Screening for Drought Tolerance in Different Sorghum

(Sorghum bicolor (L.) Moench) Varieties

Yohannes Tsago1, Mebeaselassie Andargie2*, Abuhay Takele3

1 Department of Biology, Haramaya University, P.O.Box 138, Dire Dawa, Ethiopia2 Department of Biology, Haramaya University, P.O.Box 217, Dire Dawa, Ethiopia3 Melkassa Agricultural Research Center, Nazreth, Ethiopia

E-Mail: [email protected]

Received February 15, 2013

Drought is one of the complex environmental factors affecting growth and yield of sorghum in arid and semi-arid areas of the world. Sixteen elite sorghum (Sorghum bicolor (L) Moench) genotypes were evaluated for their genetic potential to drought tolerance at callus induction and plant regeneration stage for drought tolerance. The non-ionic water soluble polymer polyethylene glycol (PEG) of molecular weight 6000 was used as osmoticum to simulate water stress. The factorial experiment was laid down in a completely randomized design which comprised of a combination of two factors (genotypes and five PEG stress level; 0, 0.5, 1.0, 1.5, and 2.0% (w/v) treatments). Data were recorded for callus induction efficiency, callus fresh weight, embryogenic callus percentage and plant regeneration percentage. Significant differences were observed among the genotypes, treatments and their interactions for the evaluated plant traits suggesting a great amount of variability for drought tolerance in sorghum. The correlation analysis also revealed strong and significant association between embryogenic callus percent and plant regeneration percent as well as between embryogenic callus percent and plant regeneration percent. By taking into consideration all the measured traits, Mann Whitney rank sum test revealed that 76T1#23 and Teshale followed by Meko, Gambella-1107 and Melkam showed better drought stress tolerance. Therefore they are recommended to be used as parents for genetic analysis, gene mapping and improvement of drought tolerance while Chelenko, Hormat and Raya appear to be drought sensitive.

Key words: callus culture, Polyethylene glycol (PEG), Sorghum bicolor L., water stress

JOURNAL OF STRESS PHYSIOLOGY & BIOCHEMISTRY Vol. 9 No. 3 2013

mailto:[email protected]

In Vitro Screening for Drought Tolerance...

ORIGINAL ARTICLE

In Vitro Screening for Drought Tolerance in Different Sorghum

(Sorghum bicolor (L.) Moench) Varieties

Yohannes Tsago1, Mebeaselassie Andargie2*, Abuhay Takele3

1 Department of Biology, Haramaya University, P.O.Box 138, Dire Dawa, Ethiopia2 Department of Biology, Haramaya University, P.O.Box 217, Dire Dawa, Ethiopia3 Melkassa Agricultural Research Center, Nazreth, Ethiopia

E-Mail: [email protected]

Received February 15, 2013

Drought is one of the complex environmental factors affecting growth and yield of sorghum in arid and semi-arid areas of the world. Sixteen elite sorghum (Sorghum bicolor (L) Moench) genotypes were evaluated for their genetic potential to drought tolerance at callus induction and plant regeneration stage for drought tolerance. The non-ionic water soluble polymer polyethylene glycol (PEG) of molecular weight 6000 was used as osmoticum to simulate water stress. The factorial experiment was laid down in a completely randomized design which comprised of a combination of two factors (genotypes and five PEG stress level; 0, 0.5, 1.0, 1.5, and 2.0% (w/v) treatments). Data were recorded for callus induction efficiency, callus fresh weight, embryogenic callus percentage and plant regeneration percentage. Significant differences were observed among the genotypes, treatments and their interactions for the evaluated plant traits suggesting a great amount of variability for drought tolerance in sorghum. The correlation analysis also revealed strong and significant association between embryogenic callus percent and plant regeneration percent as well as between embryogenic callus percent and plant regeneration percent. By taking into consideration all the measured traits, Mann Whitney rank sum test revealed that 76T1#23 and Teshale followed by Meko, Gambella-1107 and Melkam showed better drought stress tolerance. Therefore they are recommended to be used as parents for genetic analysis, gene mapping and improvement of drought tolerance while Chelenko, Hormat and Raya appear to be drought sensitive.

Key words: callus culture, Polyethylene glycol (PEG), Sorghum bicolor L., water stress

Sorghum (Sorghum bicolor (L) Moench) is a

major crop in many parts of Africa, Asia and Latin

America (ICRISAT, 2006). It is the second crop next

to maize grown across all agro-ecologies in Africa

(Wortmann et al. 2006). In Ethiopia, it is the fifth

major cereal crop in terms of area and production

next to teff (Eragrostis teff), barley (Hordeum

vulgare), wheat (Triticum aestivum) and maize (Zea

mays) (CSA 2011). Sorghum is usually grown in arid

and semi-arid parts of the tropics and sub-tropics


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mailto:[email protected]

Tsago et al.

where it is affected by drought during various

growth stages (Amjad et al. 2009). This problem is

intensified by the possible global climate change

scenarios (IPCC 2001). For example, Simon (2009)

reported that recurring drought due to deficit of

rainfall in semi-arid and dry sub-humid areas of the

country is one of the major causes of

underproduction of sorghum.

For the last half century variety development for

lowland parts of Ethiopia has focused on the

selection of early maturing varieties that can escape

drought. A number of early sorghum open-

pollinated varieties were developed and released

for these areas (Asfaw 2007). There are, however,

disadvantages to early maturity. Cultivars that

mature extremely early tend to be lower in yield

because the plants have a shorter growth period to

flower and store nutrients in the grain (Sleper and

Poehlman 2003).

On the other hand, in vitro culture studies play

tremendous role by providing efficient way of

understanding plant genetic processes in short

period of time in a controlled environment. In vitro

culture of plant cells and tissue has attracted

considerable interest over recent years because it

provides the means to study plant physiological

aspects and genetic processes in addition to

offering the potential to assist in the breeding of

improved cultivars by increasing genetic variability

(Wani et al. 2010).

For in vitro drought stress induction, one of the

most popular approaches is to use high molecular

weight osmotic substances, like PEG. PEG6000 is a

non penetrable and nontoxic osmotic substance

which is used to lower the water potential of the

culture medium and it has been used to simulate

drought stress in cultured plant tissues

(Muhammad et al. 2010). With this background, the

present study was aimed to assess the effect of PEG

induced stress on genotypic capacity of callusing,

callus growth, embryogenesis and plant

regeneration of sorghum genotypes to identify the

superior genotypes for drought tolerance.

MATERIALS AND METHODS

Plant material

The seeds of the genotypes were provided by

the National sorghum improvement project of

Melkassa agricultural research center, Ethiopia.

Sixteen sorghum varieties from more than 19

released varieties were selected based on their

agro-ecological adaptation. Five PEG concentrations

(0-2%) were used. The experiment of the study was

laid out in a completely randomized design in

factorial arrangement with three replications

(Gomez and Gomez 1976).

Callus induction under PEG stress

Murashige Skoog (MS) media (Murashige and

Skoog 1962) were prepared as described by Zhao et

al. (2010) for callus induction, pH was adjusted and

autoclaved. Thirty seeds were cultured per jar.

After four weeks of incubation, the induced calli

were excised and sub-cultured, under the same

growth conditions, and in the same MS medium to

which five level concentrations of polyethylene

glycol (PEG) (6000) (0, 0.5, 1.0, 1.5, 2.0 %) has been

added (Wani et al. 2010). The incubation period

was four weeks in which the media is replaced with

the same media in two weeks.

Plant regeneration from induced callus in PEG

stress condition

Resulting calli were excised, transferred, to jars

containing 25ml MS basal salts medium

supplemented with plant hormones and growth

regulators as described by Zhao et al. (2010) for

shoot initiation in conditions in which PEG (6000)


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(0, 0.5, 1.0, 1.5, 2.0 %) has been added. The

incubation period was four weeks.

The jars were placed in a growth chamber under

fluorescent light and at an ambient temperature of

25 ± 2ºC. The medium was changed in 15 days and

after four weeks, calli with clearly differentiated

shoots were scored as regenerating callus. Rooting

was initiated on MS medium supplemented with 3

mg l-1 indole-3-butyric acid (IBA) under the same

conditions. Regenerating calli, showing shoot and

root formations, were transferred to MS basal

medium with no phytohormones to sustain growth

of plantlets for four weeks.

Data recorded and statistical analysis

After the end of callus induction period callus

induction efficiency (CIE) was recorded as the

number of calli induced divided by total number of

seeds cultured × 100. After four weeks of culture,

callus was excised and callus fresh weight (CFW)

was determined. In the mean time, embryogenic

callus percent (ECP) was recorded as the number of

embryogenic callus obtained divided by total

number of induced callus X 100. Finally, at the end

of plant regeneration, data for plant regeneration

percent (PRP) was recorded as the number of

plantlets obtained divided by total number of

embryogenic calli cultured × 100.

Data were analyzed using SAS software 9th

Edition. The data analysis was carried out using

analysis of variance for each parameter while the

differences between treatment means was

evaluated at 1% level of significance using Fischer’s

LSD.

RESULTS AND DISCUSSION

Callus induction efficiency (CIE)

Callus induction was observed following 4 weeks

of culture. The amount and type of callus varied

with genotypes. Sorghum genotypes exhibited a

significant interaction with PEG stress levels for CIE

(Figure 1). In control (0.0% PEG), almost all explants

initiated callus. With the increasing level of PEG, the

mean percentage of callus induction reduced

significantly.

Increasing level of PEG from 0 to 2% had lowest

effect in mean CIE for genotypes Meko and Seredo

indicating their relative tolerance for drought stress

whereas the highest effect of PEG on CIE was

observed on genotype Chelenko and Raya

respectively which indicated the relative

susceptibility of these genotypes to PEG induced

stress. In Meko callus, induction percentage on

callus induction medium containing 0.5, 1.0, 1.5 and

2 g l-1 PEG was 78.9, 78.7, 78.7 and 77.8%

respectively, against 80.0% in the control set

whereas in Chelenko callus, induction percentage

on callus induction medium containing 0.5, 1.0, 1.5

and 2 g l-1 PEG was 52.2, 42.2, 36.6 and 24.4%

respectively, against 62.2% in the control set.

Generally the mean CIE decreased drastically in

genotypes under higher PEG treatments than lower

PEG treatments (Fig. 2). Decrease in CIE is a typical

response of explants of crop genotypes when

subjected to PEG stress (Matheka et al. 2008;

Biswas et al. 2002; Abdel-Ghany et al. 2004).

However, the response of CIE for PEG treatment

was genotype dependent. The difference in

decreasing trend in CIE of the genotypes might

further explain difference in osmotic regulation

among genotypes, which enables them to maintain

osmotic balance to assist initiation of callus cells

under severe stress conditions or might be due to

either water shortage which led to profuse

mutation in cellular metabolism including protein

functioning and alteration in amount of proteins


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Tsago et al.

(Plomion et al. 1999) or altered gene expression

controlling this trait (Visser 1994).

Callus Fresh Weight (CFW)

Calli were sub-cultured for a 4-week period on

MS with different levels of PEG and PGRs and fresh

weights were determined. Total callus fresh weight

varied among genotypes, as indicated on Figure 3

and in all genotypes, effect of the PEG treatments

resulted in decrease of the mean CFW with

increasing level of the PEG from 0 to 2%. Genotype

Abeshir produced the highest callus fresh weight

(345.1mg) whereas the lowest was recorded in

Birmash (315.01mg) indicating the highest damage

to callus growth in the latter genotype due to the

PEG stress.

The major effect of PEG stress in callus growth is

mainly observed in the form of decreasing the

callus fresh weight which is a typical response in

callus tissue of many crop plants (Sakthivelu et al.

2008; Lutts et al. 1996; Khalequzzaman et al. 2005;

Wani et al. 2010). Such a decrease in callus fresh

weight in response to PEG stress might be due to

water shortage which affects development and

growth of cells. Addition of PEG-6000 in solid media

lowers water potential of the medium that

adversely affect cell division leading to reduced

callus growth (Ehsanpour and Razavizadeh 2005;

Sakthivelu et al. 2008). Cell division and cell growth

are the two primary processes involved in increase

of fresh weight. In general, cell division is

considered to be less sensitive to drought when

compared with cell enlargement or growth

(Sakthivelu et al. 2008). However, both cell

expansion and cell division can be influenced by

relatively mild osmotic stress. Generally, the results

of this experiment showed that genotypic

differences in callus proliferation are genetically

determined in sorghum like other cereals.

Embryogenic Callus Percent (ECP)

More embryogenic callus (EC) generally portents

high plantlet regeneration potential, which is an

important parameter in in vitro culture systems. In

the present study, in the entire genotypes mean

ECP increased with increasing level of PEG

treatment from 0 to 1.5% and declined at 2% PEG

level (Fig.4). However, in varieties Hormat, ESH-2,

and Raya there was an overall reduction in ECP of

1.73%, 1.50% and 1.40% respectively, indicating the

highest effect of PEG treatment on ECP of these

genotypes. The highest increase in ECP was

attributed to varieties Girana-1(-3.34), Teshale (-

3.92) and Meko (-3.88), which is an indication of

lowest effect of PEG stress on embryogenic callus

formation of these genotypes.

Inconsistent variation in ECP was recorded

among genotypes in response to the five PEG stress

levels. The inconsistency could be attributed to the

difference in genotypes' strategies and responses to

the induced water deficit conditions. In line with

this, Moon and Park (2008) reported significant

increase in embryogenesis more than doubling the

average number of somatic embryos with doubling

PEG concentration. It has been reported that PEG

generally increases total soluble and reducing

sugars (Handar et al. 1983), which serve as an

osmoticum or as respiratory substrates to endure

the stress shock; inter alia, by retarding cell growth,

increasing L-proline level, and changing isozyme

profiles especially of malate dehydrogenase, acid

phosphatase, and peroxidase (Srivastava et al.

1995).

Establishment and propagation of embryogenic

callus cultures is an essential prerequisite in cereal

tissue cultures for genetic manipulation (Maggioni

et al. 1989). In the present investigation, EC

percentage decreased with increasing


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concentrations of PEG in all genotypes particularly

at the highest PEG level. It is pertinent to note that

the maximum ECP was produced in Teshale, more

than in other genotypes on stressed and non-

stressed media. Therefore this genotype may be

employed on a large scale for developing

embroygenic calluses and simultaneously to

develop drought-tolerant lines through in vitro

screening.

Plant Regeneration Percent (PRP)

Shoot regeneration after sub-culturing on fresh

MS media supplemented with different levels of

PEG started after four weeks of callus growth. The

medium was changed in 15 days and after four

weeks, calli with clearly differentiated shoots were

scored as regenerating callus, regardless of the

number of shoots. Calli regenerating plantlets were

revealed a highly significant variation among the

tested genotypes (Fig.5).

The highest effect of the PEG stress in PRP was

recorded in genotype Chelenko (26.39%) (Fig.6)

followed by Raya (23.81%) and Girana-1 (23.61%).

In Gambella-1107 (11.94%), Birmash (11.94%) and

Teshale (10.33%), PEG treatment imposed the

lowest reduction in PRP indicating that these

genotypes are the least affected by PEG treatment

in terms of plant regeneration.

The results of the present study showed that an

increment in PEG stress level caused reduction of

PRP in sorghum genotypes which is a typical

response of callus cells of many plants (Begum et al.

2011; Wani et al. 2010; Smith et al. 1985). The

typical decrease in plant regeneration in callus cells

of crop plants in response to water stress is due to

water shortage in the cells which leads to a

decrease in cell turgor and eventually cell growth.

Addition of PEG-6000 in culture media lowers water

potential of the medium that affect cell division

leading to reduced callus growth and consequently

influences regeneration (Ehsanpour and

Razavizadeh 2005; Sakthivelu et al. 2008).

Association between In vitro Traits

Callus induction efficiency had positive

significant correlation with traits such as callus fresh

weight and plant regeneration percent and callus

fresh weight had a positive significant correlation

with plant regeneration percent in addition

embryogenic callus percent showed a positive

significant correlation with plant regeneration

percent (Table 1). In contrast, callus induction

efficiency showed a significant negative correlation

with embryogenic callus percent. However, no

significant correlation was observed between callus

fresh weight and embryogenic callus percent. This

suggests that genotypes with high callus induction

also caused an increase in the number of plants

obtained from regeneration medium. The presence

of significant correlation between callus induction

efficiency and regeneration capacity of callus might

indicate that callus induction and regeneration

capacity may be controlled by the same

mechanisms.

The highly significant and strong correlation

observed between the ability of cultivars to

produce embryogenic callus and their capacity for

regeneration indicate that embryogenic callus

percentage constitute a good index for callus ability

to regenerate later on plantlets. The cultivars that

presented high embryogenic callus percentages at

the first weeks of culture have high chance to

regenerate plants after several weeks of culture.

Mann–Whitney–Wilcoxon Rank Sum Test of the

Measured Traits

To determine the most desirable drought


77

Tsago et al.

tolerant genotypes based on all traits measured,

mean rank sum method was used according to

Ezatollah et al. (2012). In this method, all the

indices, mean rank and standard deviation of ranks

of all in vitro drought tolerance criteria were

calculated and summed (RS) (Table 2). Based on this

criterion the most desirable drought tolerant

genotypes were identified.

By taking in to consideration all indices,

genotypes: Teshale (RS= 8.23) and 76T1#23 (RS=

8.39) followed by genotypes: Meko (RS= 9.29),

Gambella-1107(RS= 10.56) and Melkam (RS=11.91)

were the most drought tolerant genotypes,

respectively. Therefore they are recommended to

be used as parents for genetic analysis, gene

mapping and improvement of drought tolerance.

While genotypes: Chelenko (RS =16.36), Hormat (RS

= 15.74) and Raya (RS= 15.44) were the most

sensitive to drought, therefore they are

recommended for crossing and genetic analysis of

drought tolerance using diallel mating design.

The detection of superior genotypes Teshale,

76T1#23, Meko, Gambella-1107 and Melkam for

drought tolerance at cellular level together with

their high potential for callus induction leads to the

conclusion that a hybridization breeding procedure

using these superior plant materials supplemented

with in vitro selection for drought tolerance might

be beneficial for improving these traits in sorghum.

Hence, callus culture can be useful to speed up

sorghum improvement.

Table 1. Correlation coefficient (r) of measured traits

CIE CFW ECP PRPCIE 1CFW 0.349* 1ECPPRP

-0.125* 0.332*

0.001 0.731*

10.783* 1

CIE=Callus Induction Efficiency CFW=Callus Fresh Weight PRP=Plant Regeneration Percent

Table 2. Rank sum of measured traits

Variety CIE CFW ECP PRP Rank Mean Rank SD RS

Abshir 08 02 12 06 9.1 5.88 14.98Chelenko 15 14 03 16 10.6 5.76 16.36Raya 16 15 13 15 10.8 4.64 15.44Hormat 11 16 15 12 12.1 3.63 15.74Gubiye 05 10 07 04 08.5 3.72 12.22Gambella-1107 07 07 04 02 06.5 4.06 10.56Birmash 13 11 06 03 08.8 3.77 12.57Meko 01 01 01 05 05.0 4.29 09.29Macia 12 08 16 13 09.7 3.86 13.56Seredo 02 12 09 07 07.7 4.90 12.60Misikir 14 13 08 09 10.4 3.72 14.12Melkam 06 05 10 10 08.2 3.71 11.9176t1#23 04 04 11 08 04.7 3.53 08.39ESH-2 10 03 14 11 10.4 3.47 13.88Girana-1 09 06 02 14 08.0 5.54 13.54Teshale 03 09 05 01 05.3 3.09 08.23


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Figure 1. Mean callus induction efficiency (CIE) of genotypes for each PEG treatment level

Figure 2. Callus induction as differed across PEG treatment levels


79

Tsago et al.

Figure 3. Mean callus fresh weight of the sorghum genotypes

Figure 4. Mean embryogenic callus percent (ECP) of genotypes for each PEG treatment level

Figure 5. Mean plant regeneration percent (PRP) of genotypes for each PEG treatment level


80


Figure 6. Plant regeneration as differed across PEG treatment levels in Chelenko genotype

ACKNOWLEDGMENT

This work was financially supported by the

Ethiopian Ministry of Education which is gratefully

acknowledged. We would like to thank also

Melkassa Agricultural Research Center (MARC) for

providing the sorghum seeds.

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In Vitro Screening for Drought Tolerance in Different ... · In Vitro Screening for Drought Tolerance... ORIGINAL ARTICLE In Vitro Screening for Drought Tolerance in Different Sorghum